The detection of premalignant and malignant cells by the Papanicolaou smear (Pap smear) has greatly reduced the high mortality rate due to cervical cancer. Nevertheless, the Pap screening process is labor intensive and has remained essentially unchanged since it was first described by Papanicolaou almost 50 years ago. To perform the test, cells are exfoliated from a patient""s cervix by scraping using a spatula or brush. The scraping is then smeared on a slide, and the slide is stained and microscopically examined. The microscopic examination is a tedious process, and requires a cytotechnologist to visually scrutinize all the fields within a slide to detect the often few aberrant cells in a specimen. Consequently, the detection of abnormal specimens depends on the level of a cytotechnologist""s experience and workload, and also on the quality of the smear preparation.
A recent critical evaluation of the Pap smear reported that the error rates associated with the current technique can be startlingly high. For example, the reported false negative rate (sensitivity) ranges from 6% to 55% (see, Shingleton, H. M., et al., CA Cancer J. Clin., 45:305-320 (1995)).
As a result of these concerns, attempts have been made to automate the Pap screening process and to standardize the staining procedure. Certain of the available automated systems have been designed to improve the diagnostic yield of the Pap smear by minimizing the content of blood, mucus and other non-diagnostic debris in the examined cervical scrapings. In spite of these changes and the resulting simplification of the sample, the diagnosis of Pap smears continues to be heavily influenced by subjective bias. Thus, efforts are currently being directed towards developing alternative means of diagnosing Pap smears which are based on objective criteria such as chemical or morphological changes in cervical cells.
A number of methods have been explored to detect cytological anomalies, including those using molecular and immunological techniques. One impetus behind the development of new molecular and immunological methods is the detection of the human papilloma virus (HPV). Certain subtypes of HPV have been linked to a high incidence of abnormal lesions, and are implicated in the etiology of cervical cancer. Although these techniques are specific and detect cervical specimens at high risk, they are currently cost prohibitive and too labor intensive.
Recently, differences have been reported in the Fourier Transform Infrared (FT-IR) spectra of 156 cervical samples, of which by cytological screening, 136 were normal, 12 had cancer, and 8 had dysplasia (see, Wong et al., Proc. Natl. Acad. Sci. USA, 87:8140-8145 (1991)). This study relied on features of the mid-IR region (3000-950 cmxe2x88x921) to discriminate between the samples. The spectra of normal samples exhibited a prominent peak at 1025 cmxe2x88x921 which appears to be due to glycogen, and other less pronounced bands at 1047 cmxe2x88x921, 1082 cmxe2x88x921, 1155 cmxe2x88x921 and 1244 cm1. The spectra of specimens diagnosed with cancer exhibited significant changes in the intensity of the bands at 1025 cmxe2x88x921 and 1047 cmxe2x88x921, and demonstrated a peak at 970 cmxe2x88x921 which was absent in normal specimens. Samples with cancer also showed a significant shift in the normally appearing peaks at 1082 cmxe2x88x921, 1155 cmxe2x88x921 and 1244 cmxe2x88x921. The cervical specimens diagnosed cytologically as dysplasia exhibited spectra intermediate in appearance between normal and malignant. Based on these observations, Wong et al. concluded that FT-IR spectroscopy may provide a reliable and cost effective alternative for screening cervical specimens.
The FT-IR spectroscopic studies of Wong, et al. (1991) focused primarily on the differences between normal and malignant samples, and utilized only a few dysplastic specimens. More importantly, discrimination between specimens was achieved by inspection of spectra, and by visually detecting overt changes in peak intensity ratios at specified frequencies. Visual inspection as a basis of discrimination is not an ideal method of analysis. This approach lends itself to subjective bias and is frequently insensitive to small variations between spectra. In the case of malignant specimens, the spectral patterns are markedly altered compared to those of normal samples. However, the spectra of a great majority of specimens with low grade dysplasia (e.g. CIN I - cervical intraepithelial neoplasia) appear similar to spectra from normal samples and are difficult to distinguish. As a result, visual inspection is unreliable and unsuited for the analysis of cervical specimens.
The method of selecting peak intensity ratios to discriminate between spectra has its problems as well. This technique identifies general shapes and patterns, and like the previous approach can lack acuity in the detection of subtle differences between spectra. Other disadvantages of this method include its inability to model for interferences that can be caused by nondiagnostic debris, and/or errors that can result from sample preparation and handling techniques. Aside from the latter, this method can also fail to adequately model for baseline shifts, spectral fringes, batch to batch variations in samples and/or to account for the nonlinearities that can arise from spectroscopic instrumentation and refractive dispersion of infrared light.
More recently, others have reported a greater diversity in the spectra of specimens with dysplasia than previously reported by Wong el al. (see Morris, et al., Gynecologic Oncology 56:245-249 (1995)). Out of the 25 specimens that were evaluated, the spectra of 9/13 specimens with low grade dysplasia (CIN I) appeared essentially similar to the spectra of normal specimens. However, as dysplasia progressed from low to high (CIN I to CIN III), the magnitude of spectral differences between normal and dysplastic samples intensified. This difference was most apparent in specimens with high grade dysplasia (CIN III) which exhibited a characteristic peak at 972 cmxe2x88x921, and changes in intensity of bands at 1026 cmxe2x88x921 (decreased), 1081 cmxe2x88x921 (increased and shifted to higher frequency), 1156 cmxe2x88x921 (decreased and flattened), and 1240 cmxe2x88x921 (increased).
Even more recent studies focusing on the greater diversity in the spectra of specimens with dysplasia (Cohenford et al., Mikrochemica Acta, in press), have indicated that the extent of spectral changes could perhaps correlate with different stages of cervical abnormalities. For example, as Morris and co-workers demonstrated (Gynecologic Oncology, 56:245-249 (1995)), the spectra of specimens with severe dysplasia (CIN III) had an appearance which was intermediate between those of specimens which were diagnosed normal and those diagnosed as containing malignant cells. Unfortunately, the IR spectra of specimens which displayed mild dysplasia (CIN I) appeared essentially similar to the spectra of normal specimens.
The progression of dysplastic cells to malignant cells is not only well documented, but is also of fundamental importance in early diagnosis and prevention of cancer. As it is important, from a clinical point of view, to distinguish those specimens with dysplastic cells from those with only normal cells, a generally useful method using IR spectroscopy must be capable of this rather fine distinction. Quite surprisingly, the present invention provides such methods.
The present invention provides methods for the spectroscopic detection of chemical differences between cells in a sample of cells or tissue. The methods provided include single-cell infrared microspectroscopy and infrared spectroscopic imaging. In general, the data acquired by the microspectroscopic or imaging process is analyzed using multivariate analysis. In certain embodiments the detected chemical difference is associated with a disease state, particularly a malignant or premalignant state. In these embodiments, the invention provides a method for the early detection of a malignant or premalignant state. In another aspect, the invention provides a spectroscopic method for distinguishing between cells in a cell sample which are cytologically and chemically xe2x80x9cnormalxe2x80x9d and those cells in a cell sample which appear cytologically normal but which are, in fact, xe2x80x9cchemically aberrant.xe2x80x9d In yet another aspect, the invention provides a spectroscopic method for the identification of women who are at high risk for developing cervical dysplasia.
Throughout the discussion below and in the examples cervical cells are utilized as an exemplary cell type. The exemplary use of cervical cells does not limit or define the scope of the present invention.
Thus, in a first aspect, the invention provides a method for detecting chemical differences between a cell sample and a reference cell sample, utilizing single-cell infrared microspectroscopy, the method comprising:
(a) directing a beam of infrared light at single cells in a cell sample to produce absorption data for each of the single cells;
(b) comparing the absorption data for each of the single cells with infrared absorption spectra acquired from individual cells in at least one reference cell sample to provide comparison data for each of the single cells in the cell sample.
In a second aspect, the invention provides a method for detecting chemical differences between a cell sample and a reference cell sample utilizing single-cell infrared microspectroscopy, the method comprising:
(a) directing a beam of infrared light at single cells in the cell sample to produce absorption data for each of the single cells;
(b) comparing the absorption data for the single cells with infrared absorption spectra acquired from individual cells in at least one reference cell sample to provide comparison data for the single cells;
(c) generating scores for the absorption data using the comparison data;
(d) establishing a mean of the scores for the single cells;
(e) comparing the mean with a normal distribution curve of scores, to detect the chemical differences in the cell sample.
In a third aspect, the invention is a method for detecting chemical differences between a cell sample and a reference cell sample utilizing infrared spectroscopic imaging, the method comprising:
(a) directing a beam of infrared light at a cell sample to produce absorption data, substantially simultaneously, for each of a plurality of single cells in the cell sample;
(b) comparing the absorption data for each of the single cells with a reference set of absorption spectra acquired from individual cells in at least one reference cell sample to provide comparison data for each of the single cells in the cell sample.
In a fourth aspect, the invention is a method for identifying women at high risk for developing cervical dysplasia utilizing single-cell infrared microspectroscopy, the method comprising:
(a) directing a beam of infrared light at single cells in a cervical cell sample to produce absorption data for each of the single cells;
(b) comparing the absorption data from the diagnostic cervical cell sample with a reference set of absorption spectra to identify the woman at high risk for developing cervical dysplasia, the reference set comprising;
absorption spectra from individual cervical cells in cervical cell reference samples taken from women having no history of dysplasia, each of the reference samples having a combination of individual cells exhibiting at least one first spectrum pattern and at least one second spectrum pattern differing from each other in either source or pattern.